Rescuing Endothelial Barrier Dysfunction in Sepsis: miR-23b-3p Antagonizes SMAD3-Mediated EndoMT and Vascular Hyperpermeability

Abstract

Background Sepsis-associated acute lung injury (ALI) is driven by endothelial barrier dysfunction and endothelial–mesenchymal transition (EndoMT), mediated by TGF-β1/SMAD3 signaling. Despite the therapeutic potential of SMAD3, current inhibitors face limitations. As endogenous small molecules that are closely related to physiological regulatory processes, miRNAs have more potential research value for regulating SMAD3. Methods Screening multiple databases revealed that miR-23b-3p was the sole miRNA targeting SMAD3. LPS-stimulated HUVECs and cecal ligation/puncture (CLP) mice were used to model sepsis. Lentivirus was used to construct stable strains. The functional performance and mechanism were verified by key techniques, including dual-luciferase assays, rescue experiments, RT–qPCR/Western blotting, monocyte adhesion/permeability assays, and histopathology. Results In LPS-stimulated HUVECs, miR-23b-3p downregulation correlated with TGF-β1/SMAD3 activation, EndoMT progression, and barrier disruption. miR-23b-3p overexpression reversed these effects by restoring the expression of junctional proteins and suppressing the expression of mesenchymal markers. ChIRP-qPCR, RNA-pull-down, and dual-luciferase assays confirmed the direct miR-23b-3p—SMAD3 3’UTR interaction. Rescue experiments demonstrated that miR-23b-3p counteracts TGF-β1/SMAD3 hyperactivation. In CLP mice, intratracheal agomiR-23b-3p attenuated lung injury, normalized alveolar architecture, and reduced vascular leakage by suppressing endothelial Smad3 upregulation. Conclusion miR-23b-3p is a SMAD3-targeting regulator that inhibits EndoMT and repairs endothelial barrier integrity. Mechanistically, miR-23b-3p preserves endothelial homeostasis via SMAD3-dependent EndoMT inhibition. This study provides mechanistic insights and a miRNA-based therapeutic strategy for sepsis-induced ALI.